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Fragment-based drug discovery (FBDD) has emerged as a powerful strategy to confront the challenges faced by conventional drug development approaches, particularly in the context of central nervous system (CNS) disorders. FBDD involves the screening of libraries that comprise thousands of small molecular fragments, each no greater than 300 Da in size. Unlike the generally larger molecules from high-throughput screening that limit customisation, fragments offer a more strategic starting point. These fragments are inherently compact, providing a strong foundation with good binding affinity for the development of drug candidates. The minimal elaboration required to transition the hit into a drug-like molecule is not only accelerated, but also it allows for precise modifications to enhance both their activity and pharmacokinetic properties. This shift towards a fragment-centric approach has seen commercial success and holds considerable promise in the continued streamlining of the drug discovery and development process. In this review, we highlight how FBDD can be integrated into the CNS drug discovery process to enhance the exploration of a target. Furthermore, we provide recent examples where FBDD has been an integral component in CNS drug discovery programs, enabling the improvement of pharmacokinetic properties that have previously proven challenging. The FBDD optimisation process provides a systematic approach to explore this vast chemical space, facilitating the discovery and design of compounds piece by piece that are capable of modulating crucial CNS targets. [ABSTRACT FROM AUTHOR]

Biological membranes consist of integral and peripheral protein-associated lipid bilayers. Although constituent lipids vary among cells, membrane lipids are mainly classified as phospholipids, glycolipids, and sterols. Phospholipids are further divided into glycerophospholipids and sphingophospholipids, whereas glycolipids are further classified as glyceroglycolipids and sphingoglycolipids. Both glycerophospholipids and glyceroglycolipids contain diacylglycerol as the common backbone, but their head groups differ. Most glycerolipids have polar head groups containing phosphate esters or sugar moieties. However, trace components termed glycero-glycophospholipids, each possessing both a phosphate ester and a sugar moiety, exist in membranes. Recently, the unique biological activities of glycero-glycophospholipids have attracted considerable attention. In this review, we describe the structure, distribution, function, biosynthesis, and chemical synthetic approaches of representative glycero- glycophospholipids -- phosphatidylglucoside (PtdGlc) and enterobacterial common antigen (ECA). In addition, we introduce our recent studies on the rare glycero-glyco"pyrophospho"lipid, membrane protein integrase (MPIase), which is involved in protein translocation across biomembranes. [ABSTRACT FROM AUTHOR]

Chemical proteomics, which involves studying the covalent modifications of proteins by small molecules, has significantly contributed to our understanding of protein function and has become an essential tool in drug discovery. Mass spectrometry (MS) is the primary method for identifying and quantifying protein‐small molecule adducts. In this review, we discuss various methods for measuring proteomic reactivity using MS and covalent proteomics probes that engage through reactivity‐driven and proximity‐driven mechanisms. We highlight the applications of these methods and probes in live‐cell measurements, drug target identification and validation, and characterizing protein‐small molecule interactions. We conclude the review with current developments and future opportunities in the field, providing our perspectives on analytical considerations for MS‐based analysis of the proteomic reactivity landscape. [ABSTRACT FROM AUTHOR]

A novel transformation of the multiplexing potential to the throughput potential of multiple reaction monitoring mass spectrometry is presented for targeted quantitation of proteins. Herein, this method is termed as ultrathroughput multiple reaction monitoring (UMRM) mass spectrometry. It integrates the use of stable isotope dilution-multiple reaction monitoring mass spectrometry and peptide derivatization with inexpensive and commercial chemicals. One-experiment quantitation of a common signature peptide in 25 different samples demonstrates proof-of-concept for the unprecedented throughput potential of the UMRM technology. 10.1021/ac9026274

Multiple reaction monitoring tandem mass spectrometry becomes an important strategy for measuring protein targets in complex biomatrixes. Active chemical modification of peptides like phenylthiocarbamoylation has unique potential for improving the measurement. This potential is enabled by active participation of a modifying group in site-preferential dissociation of modified peptides, which produces certain fragment ions at very high yields and in a sequence-independent manner. In this work, a novel combination of energy-resolved mass spectrometry with substituent effect investigation is used to analyze important factors that control the specificity of the site-preferential dissociation of phenylthiocarbamoyl peptides. On the basis of the linear correlation between collision energy and the Hammett constant as well as computational studies, it is found that the initial enhanced capture of a mobile proton and the subsequent, site-directed intramolecular proton transfer are important to the high yields (~70-90%) for producing two types of fragment ions of phenylthiocarbamoyl peptides: the modified [b.sub.1] ion and the complementary [y.sub.n-1] ion. This understanding will help the design of new modification reagents. When integrated with the throughput and the signal-enhancing potential of peptide modification, active chemical modification of peptides will significantly advance mass spectrometry-based, targeted proteome analysis. 10.1021/ac902120k

The exact mass of a peptide differs characteristically from its nominal mass by a value called the mass defect. Limited by possible elemental compositions, the mass defect of peptides has a restricted range, resulting in an unoccupied mass spectral space in every mass-to-charge unit. The method of fragment ion mass defect labeling (FIMDL) places characteristic fragment ions of modified peptides as reporters into unused spectral space where no native peptide fragment ions exist. In this labeling method, peptides are chemically modified in solution and the modified peptides, upon gas-phase collision in a mass spectrometer, generate fragment ions with significantly shifted mass defects. In this work, the efficiency of iodine stable isotope-containing reagents for shifting mass defects of peptide fragment ions was systematically investigated, through derivatization of peptide N-termini with various reagents containing one or more chlorine, bromine, or iodine atoms. The observed efficiency for the iodine atom placing the labeled fragment ions into unoccupied spectral space agreed well with theoretical predictions from averagine-scaling analysis of ion masses. On the basis of the gas-phase stability of different labeling groups and their involvement in collisional dissociation of modified peptides, peptide modifications were classified into three categories: passive, type I active, and type II active. Each modification type has its unique potential in different proteome analyses. Possible proteomics applications of FIMDL are discussed and compared with proteome analyses currently being practiced in the field. Principles obtained from this survey study will provide a guideline in developing novel FIMDL reagents for advanced proteomics analysis.

A novel method is reported to modify the phosphate groups on phosphoserine peptides to the corresponding phosphoramidates, using 2-aminobenzylamine. Upon collision-induced dissociation, the modified peptides release the positively charged phosphoramidate that via gas-phase intramolecular elimination forms a cyclophosphoramidate (CyPAA) ion, the protonated form of 1,4-dihydro-2-hydroxy-2-oxobenzo[3,1,2]oxazaphosphorine. The positive nature of the ion eliminates the need for real-time instrument polarity switching and greatly increases the versatility of commonly used mass spectrometers for phosphopeptide analysis. This ion has sufficient mass defect, due to containing a phosphorus atom and a high content of oxygen atoms, which makes mass spectrometers of medium mass resolution and accuracy adequate for separating the ion from isobaric interfering ions. The specificity of the CyPAA ion for detecting phosphoserine peptides in complex peptide mixtures is comparable to the specificity of the phosphotyrosine immonium ion for phosphotyrosine peptides, allowing the efficient data complexity reduction for fast and focused analysis of phosphoserine-containing peptides.

Abstract: Multiple reaction monitoring mass spectrometry coupled with stable isotope dilution has become the method of choice for quantifying target proteins in complex biological samples. It quantifies signature peptides based on the sequential detection of peptide precursor ions and their fragments generated upon gas-phase collision. Heavy-isotope (13C and/or 15N) labeled peptides or proteomes are commonly used as internal reference standards for quantitation. However, use of these standards is becoming expensive when large numbers/amounts of reference peptides are needed. The search for low-cost, labeled references for proteomic quantitation such as those with 2H-labels is an object of constant pursuit. In order to take the cost advantage of 2H-labels, this work examines whether or not the known chromatographic separation of 2H-based peptides from the native counterparts affects the peptide quantitation using multiple reaction monitoring mass spectrometry. Experimental results from model peptides and proteome digests indicate that targeted mass spectrometry quantitation of the 2H- and 13C/15N-based peptides are comparable. [Copyright &y& Elsevier]

A novel transformation of the multiplexing potential to the throughput potential of multiple reaction monitoring mass spectrometry is presented for targeted quantitation of proteins. Herein, this method is termed as ultrathrough-put multiple reaction monitoring (UMRM) mass spectrometry. It integrates the use of stable isotope dilution-multiple reaction monitoring mass spectrometry and peptide derivatization with inexpensive and commercial chemicals. One-experiment quantitation of a common signature peptide in 25 different samples demonstrates proof-of-concept for the unprecedented throughput potential of the UMRM technology. [ABSTRACT FROM AUTHOR]

The exact mass of a peptide differs characteristically from its nominal mass by a value called the mass defect. limited by possible elemental compositions, the mass defect of peptides has a restricted range, resulting in an unoccupied mass spectral space in every mass-to-charge unit. The method of fragment ion mass defect labeling (FIMDL) places characteristic fragment ions of modified peptides as reporters into unused spectral space where no native peptide fragment ions exist. In this labeling method, peptides are chemically modified in solution and the modified peptides, upon gas-phase collision in a mass spectrometer, generate fragment ions with significantly shifted mass defects. In this work, the efficiency of iodine stable isotope-containing reagents for shifting mass defects of peptide fragment ions was systematically investigated, through derivatization of peptide N-termini with various reagents containing one or more chlorine, bromine, or iodine atoms. The observed efficiency for the iodine atom placing the labeled fragment ions into unoccupied spectral space agreed well with theoretical predictions from averagine-scaling analysis of ion masses. On the basis of the gas-phase stability of different labeling groups and their involvement in collisional dissociation of modified peptides, peptide modifications were classified into three categories: passive, type I active, and type 11 active. Each modification type has its unique potential in different proteome analyses. Possible proteomics applications of FLMDL are discussed and compared with proteome analyses currently being practiced in the field. Principles obtained from this survey study will provide a guideline in developing novel FIMDL reagents for advanced proteomics analysis. [ABSTRACT FROM AUTHOR]

A novel method is reported to modify the phosphate groups on phosphoserine peptides to the corresponding phosphoramidates, using 2-aininobenzylamine. Upon collision-induced dissociation, the modified peptides release the positively charged phosphoramidate that via gas-phase intramolecular elimination forms a cyclophosphoramidate (CyPAA) ion, the protonated form of 1,4-dihydro-2hydroxy-2-oxobenzo[3, 1 ,2]oxazaphosphorine. The positive nature of the ion eliminates the need for real-time instrument polarity switching and greatly increases the versatility of commonly used mass spectrometers for phosphopeptide analysis. This ion has sufficient mass defect, due to containing a phosphorus atom and a high content of oxygen atoms, which makes mass spectrometers of medium mass resolution and accuracy adequate for separating the ion from isobaric interfering ions. The specificity of the CyPAA ion for detecting phosphoserine peptides in complex peptide mixtures is comparable to the specificity of the phosphotyrosine immonium ion for phosphotyrosine peptides, allowing the efficient data complexity reduction for fast and focused analysis of phosphoserine-containing peptides. [ABSTRACT FROM AUTHOR]